Abstract 177: The Effects of Inflammation, Hyperglycemia and Cyclic Strain on Osteoprotegerin Production in Human Aortic Smooth Muscle Cells

Abstract

Introduction: A pivotal step in vascular calcification is the transformation of smooth muscle cells (SMCs) to osteoblast-like cells. Osteoprotegerin (OPG), a soluble protein produced by SMCs, inhibits this transformation and may represent a therapeutic target for calcification reduction. Hyperglycemia, inflammation and increased levels of cyclic strain are all important inducers of calcification in vivo. The purpose of this study was to determine the effects of these stimuli, both alone and in combination, on OPG production by human aortic SMCs (HASMCs) in vitro. Methods: HASMCs were initially exposed to a single stimulus, with OPG levels in cell-conditioned media measured at 24 and 48 hours by ELISA (n=6 for all experiments). Equibiaxial cyclic strain was produced using a Flexercell® Tension-Plus FX-4000T system (0 or 12.5%), whilst hyperglycemic and inflammatory conditions were induced using glucose (6, 15 and 30 mmol/l) and tissue necrosis factor alpha (TNFα, 10 and 100 ng/ml) respectively. HASMCs were then exposed to a combination of hyperglycemia (15 mmol/l), inflammation (10 ng/ml TNFα) and strain (10%), after which levels of OPG and runt-related transcription factor 2 (RUNX2, a marker of osteoblastic transformation) were assessed via ELISA and qRT-PCR, respectively. Results: High levels of cyclic strain (12.5%) significantly increased OPG levels in the cell-conditioned media at 24 and 48 hours. OPG levels were also dose-dependently increased by treatment with TNFα at 24 and 48 hours. At 24 hours with 15 and 30mmol/l glucose, there was no change in OPG levels, but at 48 hours increased OPG levels were observed at both concentrations. In the combination experiment, OPG levels decreased, while RUNX2 mRNA levels increased, after 48 hrs. Conclusions: Cyclic strain, hyperglycemia, and TNFα increased OPG release by HASMCs when administered in isolation. Applied in combination however, these stimuli decreased OPG levels in parallel with an increase in a marker of osteoblastic transformation. This data suggests that inhibition of vascular calcification by OPG may be actively down-regulated when multiple pro-calcification stimuli co-exist, and supports further investigation of OPG as a therapeutic target for calcification reduction.